Immunocapture-based measurements of mammalian pyruvate dehydrogenase complex

ABSTRACT

The present invention is based on the discovery that an antibody specific for PDH complex can be used to immunoprecitate PDH complex from the patient sample in an active state. Therefore the anti-PDH complex specific antibody can be used to determine the amount of and/or active state of PDH in a patient sample. The invention immunoassay methods for determining the amount and/or active state of PDH complex present in a patient sample are useful for screening to identify individuals having symptoms indicating malfunction of PDH complex. For example, the invention methods can be used to screen individuals for symptoms of onset of the diabetic state, such as insulitis, and for diagnosing late onset diseases, such as diabetes, Alzheimer&#39;s and the like.

This invention relates to immunoassays, in particular, to immunoassaysfor determining disorders of mitochondrial energy metabolism anddiseases associated with late onset mitochondrial disorders.

BACKGROUND OF THE INVENTION

The bulk of ATP used by many cells to maintain homeostasis is producedby the oxidation of pyruvate in the TCA cycle. During this oxidationprocess, reduced nicotinamide adenine dinucleotide (NADH) and reducedflavin adenine dinucleotide (FADH₂) are generated. The NADH and FADH₂are principally used to drive the processes of oxidativephosphorylation, which are responsible for converting the reducingpotential of NADH and FADH₂ to the high energy phosphate in ATP.

The fate of pyruvate depends on the cell energy charge. In cells ortissues with a high energy charge, pyruvate is directed towardgluconeogenesis, but when the energy charge is low pyruvate ispreferentially oxidized to CO₂ and H₂O in the TCA cycle, with generationof 15 equivalents of ATP per pyruvate. The enzymatic activities of theTCA cycle (and of oxidative phosphorylation) are located in themitochondrion. When transported into the mitochondrion, pyruvateencounters two principal metabolizing enzymes: pyruvate carboxylase (agluconeogenic enzyme) and pyruvate dehydrogenase (PDH), the first enzymeof the PDH complex. With a high cell-energy charge coenzyme A (CoA) ishighly acylated, principally as acetyl-CoA, and able allosterically toactivate pyruvate carboxylase, directing pyruvate towardgluconeogenesis. When the energy charge is low CoA is not acylated,pyruvate carboxylase is inactive, and pyruvate is preferentiallymetabolized via the PDH complex and the enzymes of the TCA cycle to CO₂and H₂O. Reduced NADH and FADH₂ generated during the oxidative reactionscan then be used to drive ATP synthesis via oxidative phosphorylation.

Recently there has been renewed interest in the structure andfunctioning of the pyruvate dehydrogenase (PDH) complex, due torealization that altered PDH complex activity is a feature of many humandisorders ranging from the relatively uncommon primary PDH deficiency[1] to major causes of morbidity and mortality, such as diabetes,starvation, sepsis and Alzheimer's disease [2-7]. PDH is a mitochondrialenzyme central to aerobic carbohydrate metabolism. It catalyzes theirreversible decarboxylation of pyruvate in the presence of CoA and NAD+to generate CO₂, acetyl CoA and NADH [8]. PDH is one of the largestenzymes known (MW around 8,000,000) and consists of several components,each present in the complex in multiple copies. Catalytic functioninvolves a pyruvate dehydrogenase (E1), which contains E1α and E1βsubunits, dihydrolipoamide transacetylase (E2), and dihydrolipoamidedehydrogenase (E3) [8-10]. One structural subunit of the PDH complex hasbeen described, the E3 binding protein (E3B P), which contributes to theproper assembly of the complex by promoting the interaction between E2and E3 components [10].

More particularly, the PDH complex is comprised of multiple copies of 3separate enzymes: pyruvate dehydrogenase (20-30 copies), dihydrolipoyltransacetylase (60 copies) and dihydrolipoyl dehydrogenase (6 copies).The complex also requires 5 different coenzymes: CoA, NAD⁺, FAD⁺, lipoicacid and thiamine pyrophosphate (TPP). Three of the coenzymes of thecomplex are tightly bound to enzymes of the complex (TPP, lipoic acidand FAD⁺) and two are employed as carriers of the products of PDHcomplex activity (CoA and NAD⁺).

The first enzyme of the complex is PDH itself, which oxidativelydecarboxylates pyruvate. During the course of the reaction, the acetylgroup derived from decarboxylation of pyruvate is bound to TPP. The nextreaction of the complex is the transfer of the 2-carbon acetyl groupfrom acetyl-TPP to lipoic acid, the covalently bound coenzyme of lipoyltransacetylase. The transfer of the acetyl group from acyl-lipoamide toCoA results in the formation of 2 sulfhydryl (SH) groups in lipoate,requiring reoxidation to the disulfide (S—S) form to regenerate lipoateas a competent acyl acceptor. The enzyme dihydrolipoyl dehydrogenase,with FAD⁺ as a cofactor, catalyzes that oxidation reaction. The finalactivity of the PDH complex is the transfer of reducing equivalents fromthe FADH₂ of dihydrolipoyl dehydrogenase to NAD⁺. The fate of NADH isoxidation via mitochondrial electron transport to produce 3 equivalentsof ATP.

The reactions of the PDH complex serve to interconnect the metabolicpathways of glycolysis, gluconeogenesis and fatty acid synthesis to theTCA cycle. As a consequence, activity of the PDH complex is highlyregulated by a variety of allosteric effectors and by covalentmodification. Importance of the PDH complex to the maintenance ofhomeostasis is evident from the fact that, although diseases associatedwith deficiencies of the PDH complex have been observed, affectedindividuals often do not survive to maturity. Primary PDH deficiency isa severe disorder, affecting different tissues [21, 22]. Mutations inthe E1α, E2, E3BP subunits and in PDP have been reported in PDHdeficient patients [1, 23-25], but around 95% of all mutations are inthe E1α subunit of the complex. The diagnosis of PDH deficienciesremains difficult. Recently an immunocytochemical method to aid initialdiagnosis of these disorders has been discovered [26]. By this approach,mutations that alter expression of the E1α subunit, which is an x-linkedgene, can be detected even when present in only 1-2% of cells, as insome female carriers, who are most often mosaic for the mutation.

Since the energy metabolism of highly aerobic tissues, such as thebrain, is dependent on normal conversion of pyruvate to acetyl-CoA,aerobic tissues are most sensitive to deficiencies in components of thePDH complex. Most genetic diseases associated with PDH complexdeficiency are due to mutations in PDH. The main pathologic result ofsuch mutations is moderate to severe cerebral lactic acidosis andencephalopathies.

In addition, recent studies have shown that there is evidence of a roleof mitochondrial dysfunction as a direct cause of and/or as acomplication of a number of late-onset diseases. Alteration of OXPHOSfunctioning due to reduced synthesis and/or posttranslationalmodification of component proteins (and mtDNA) is now thought to be amajor contributor to Parkinson's disease, Huntington's disease,Alzheimer's disease, Downs Syndrome, Schizophrenia, late-onset type IIdiabetes (also called NIDDM), and even the aging process itselfMoreover, altered OXPHOS can also be an unintended consequence andcomplication of the treatment of human diseases; for example,reperfusion injury is a problem for heart attack victims and a criticalissue in all organ transplants. Re-oxygenation of tissue that has becomeanaerobic by a cut-off of blood supply produces high concentrations oftoxic-free radicals as this strong oxidant reacts with the highlyreduced OXPHOS proteins, and it is this process that is thought to killcells. Therapy for HIV infection with nucleoside reverse transcriptaseinhibitors, such as AZT and DDC, causes a myopathy and lipidopathy inmany patients due to a loss of oxidative OXPHOS function resulting fromthe reduction of mitochondrial protein synthesis. The myopathy that isan occasional side effect of statin use to treat hypercholesterolemiahas also been attributed to mitochondrial toxicity of these drugs. Ageneral discussion of research into the molecular bases ofmitochondrially-related health disorders is found in Lib et al. (2002)Journal of Histochemistry and Cytochemistry 50:877-884 and Hanson et al.(2002) Journal of Histochemistry and Cytochemistry 50: 1281-288.

Therefore, there is a need in the art for new and better methods formeasuring the amount and active state of PDH complex in patient samples,particularly in a format that is compatible with high throughputscreening techniques.

SUMMARY OF THE INVENTION

The present invention is predicated on the discovery that an antibodyspecific for PDH complex can be used to immunoprecitate PDH complex froma patient sample in an active state. Therefore the anti-PDH complexspecific antibody can be used to determine the amount of and/or activestate of PDH in a patient sample. The invention immunoassay methods fordetermining the amount and/or active state of PDH complex present in apatient sample are useful for screening to identify individuals havingsymptoms indicating malfunction of PDH complex. For example, theinvention methods can be used to screen individuals for symptoms ofonset of the diabetic state, such as insulitis, and for diagnosing lateonset diseases, such as diabetes, Alzheimer's and the like.

Accordingly, in one embodiment the invention provides methods fordetermining the amount of pyruvate dehydrogenase (PDH) complex in abiological sample by contacting a sample comprising PDH complex with anisolated antibody that specifically binds to PDH complex underconditions to allow specific binding of the antibody to solubilized PDHcomplex present in the sample to form an immunocomplex. Theimmunocomplex is separated from remaining sample contents and the amountof the PDH complex in the separated immunocomplex is determined, therebydetermining the amount of the PDH complex in the patient sample.

In another embodiment, the invention provides methods for measuringactivity of PDH complex in a sample. In this procedure a samplecomprising PDH complex is contacted with an isolated antibody thatspecifically binds to PDH complex under conditions to allow formation ofan immunocomplex of the antibody and the PDH complex present in thesample and the immunocomplex is contacted with a reaction mixturecomprising a non-limiting amount of substrates necessary for activity ofthe PDH complex. Detection of the amount of NADH produced in thereaction mixture indicates the active state of the PDH complex in thesample.

In yet another embodiment, the invention provides methods fordetermining the level of activity of PDH complex in a sample bymeasuring its level of phosphorylation. In this embodiment of theinvention methods, an immunocomplex is formed as described herein andcontacted with a reaction mixture comprising a non-limiting amount ofsubstrates necessary for activity of the PDH complex. The remainingsample contents are separated from the immunocomplex; and the level ofphosphorylation of immunocomplexed PDH complex in the in the sample isdetected and compared with that of an unphosphorylated PDH complexstandard. A level of phosphorylation greater than that in the standardindicates a lowered level of activity, and a level of phosphorylationsubstantially equal to that of the PDH complex in the sample indicates anormal level of activity of the PDH complex in the sample.

In still another embodiment, the invention provides methods forscreening to detect an active agent that modifies inhibitor or activatoractivity of a known inhibitor or activator of PDH complex. A samplecontaining PDH complex in the presence of a known inhibitor or activatorand a test active agent is contacted with a PDH compleximmunoprecipitating antibody under conditions that allow formation of anantibody/PDH complex immunocomplex. The degree to which the test activeagent modifies the inhibitor or activator activity of the knowninhibitor or activator in the sample is detetected and compared toinhibitor or activator activity of the known inhibitor or activator inthe absence of the test active agent, thereby indicating the degree towhich inhibitor or activator activity is modified by the test activeagent.

In a still further embodiment, the invention provides methods forscreening patients to identify those suspected of having a late onsetmitochondrial disorder by performing the invention immunoassay asdescribed herein so as to detect a decrease in the amount or activestate of PDH complex in a patient sample as compared with an amount oractive state of PDH complex in a corresponding normal sample. A detecteddecrease indicates the patient is suspected of having a late onsetmitochondrial disorder, such as late onset diabetes, Huntington's,Parkinson's or Alzheimer's disease, ALS (amyotrophic lateral sclerosis),or Schizophrenia.

In another embodiment, the invention provides kits containing one ormore anti-PDH monoclonal antibodies that are useful for conducting theinvention immunoassays.

A BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a graph showing activity of immunocaptured PDH as measured inmoles NADH produced per 60 min. Activity was not detected when one ofthe substrates for the reaction was omitted. Control reaction containsthe “full” reaction mixture as described in the Examples, while otherslack one of the substrates or co-factors of the reaction as indicated:−CoA=minus CoA; −pyruvate=minus pyruvate; NAD=minus NAD⁺; TPP=minusthiamine pyrophosphate; control=in the presence of NAD⁺, pyruvate, CoA,TPP, MgCl₂ and cysteine as reducing agent.

FIG. 2 is a graph summarizing results of quantitation assays ofsaturation binding of various sized samples of human heart PDH tomicrotiter plates. Solubilized human heart mitochondria (HHM) wasincubated with antibodies bound to 96-well microplate. After one hour ofincubation, the supernatant was collected and applied to a secondantibody-coated plate. After an additional one hour of incubation, theactivities of PDH (residual activity) were detected and compared withthose of the first plate (activity measured) for the various-sizedsamples.

FIG. 3 is a graph summarizing the results of tests for specificinhibition and activation of immunocaptured PDH activity. Knowninhibitors and activators as shown were added directly to wells ofmicrotiter plates and incubated with already immunocaptured PDH.Activity of the complex was monitored by measuring production of NADHper min/mg mitochondrial protein. Unmodified activity of human heart PDH(control) was set to 100%.

FIG. 4 is a graph showing the results of analysis of PDH-deficient celllines MRC5, 404, 581 and 594 by the immunocapture microplate-based PDHactivity assay. Pyruvate-dependent NADH production was followedfluorometrically in the presence of resazurin and diaphorase using anexcitation wavelength of 530 nM and an emission wavelength of 590 nM.PDH activities in patient fibroblasts were calculated in relation toMRC5 control fibroblasts, where activity was set to 100%.

DETAILED DESCRIPTION OF THE INVENTION

The present invention is predicated on the discovery that some of themAbs used for detection of PDH subunits also immunoprecipitate the boundenzyme. Functioning of the bound enzyme was measured bothspectrophotometrically or by a coupled fluorescent assay for NADHproduction in a high-throughput format, which is useful not only foranalyzing patient samples but in screening for PDH-active drugs andenvironmental toxins.

The invention provides a micro scale method for the immunocapture andfunctional detection of active mitochondrial PDH complex fromsolubilized human mitochondria using a specific anti-human E-2monoclonal antibody in an immunocapture format. The assay is suitablefor high-throughput screening of samples containing mitochondria, forexample obtained from human heart, human brain, human culturedfibroblast or bovine heart. The assay is specific because irrelevantantibodies fail to capture detectable activity. The assay is alsoquantitative and can be used to measure the amount of solubilizedmitochondrial F1/F0 ATPase in samples relative to a reference controlcontaining a known amount of PDH complex, for example. Thus, theinvention assay can be used to detect disorders in production and/orutilization of PDH complex in patient samples.

The invention assays are also sensitive, requiring as little as 10nanograms of mitochondrial protein per test, and have a wide dynamicrange of at least 1000-fold. For example, when human heart mitochondriaare used as a target, the assay is quantitative over a range from 10nanograms to 10 micrograms of mitochondrial protein per sample. Thus theinvention methods are suitable for use in high-throughput screeningassay formats.

In one embodiment, the invention provides methods for determining theamount of pyruvate dehydrogenase (PDH) complex in a biological samplecomprising providing a solid support having attached thereto antibodiesspecific for PDH complex; contacting the antibodies attached to thesolid support with a sample comprising solubilized human fibroblasts,human heart mitochondria or bovine heart mitochondria so that theantibodies immunocapture any PDH complex present in the sample;separating remaining sample contents from the solid support; anddetecting the amount of immunocaptured PDH complex on the solid support.For example, antibodies are anti-E2 specific antibodies.

In another embodiment, the invention provides methods for measuringactivity of PDH complex in a sample by contacting PDH complex bound to asolid support via an antibody specific for PDH complex with a samplecomprising solubilized human fibroblasts, human heart mitochondria orbovine heart mitochondria so that the antibodies immunocapture any PDHcomplex present in the sample. The immunocaptured PDH complex is thencontacted with a reaction mixture comprising a non-limiting amount ofone or more substrates necessary for activity of the PDH complex; andthe amount of NADH produced in the reaction mixture is detected, whereinthe amount of NADH produced indicates efficacy of biological function ofthe PDH complex. The detection of NADH production can be convenientlymeasured by transferring an electron from the reduced NADH to anelectron acceptor molecule, such as an electron acceptor dye molecule toproduce a change in signal from the molecule, such as absorbance of theelectron acceptor dye molecule. The reaction mixture is then monitoredto detect the change in signal; wherein the magnitude of the changeindicates the biological activity of the PDH complex

The invention provides methods for the immunocapture and functionaldetection of active mitochondrial PDH complex from solubilized human orbovine mitochondria or from using a specific anti-E2 specific monoclonalantibody in an immunocapture format. The antibodies can be attached to asolid support and the assay can be conducted using high-throughputscreening of samples containing mitochondria, for example obtained fromhuman heart, human brain, human cultured fibroblast or bovine heart. Theassay is specific for PDH complex, as the antibody does not capturesubunits of the enzyme or 2-oxo-acid dehydrogenase

A solid support, such as a 96 well microtiter plate, coated withmonoclonal anti-PDH-E2 subunit, can be used to coat the solid supportwith PDH for conducting activity studies. By measuring NADH productionwhen the plate is contacted with a suitable reaction mixture containingrequired substrates. The assay is also quantitative and can be used tomeasure the amount of activity, for example on the basis of nmolNADH/min/mg of mitochondrial protein, by comparison to a referencecontrol containing a known amount of PDH. Thus, the invention assay canbe used to detect disorders in production of PDH in patient samples.

The invention assays are also sensitive, requiring as little as 50micrograms of mitochondrial protein to saturate a 96-well microplate andhave a wide dynamic range. Thus the invention methods are suitable foruse in high-throughput screening assay formats.

In another embodiment, the invention PDH functional immunocapture assayis suitable for use as a diagnostic assay to detect any type ofactivity-affecting defect of mitochondrial PDH complex in humans, suchas catalytic defects, the presence or absence of subunit antigen,defects in assembly of the enzyme complex, and the like.

In yet another embodiment of the invention, the degree ofphosphorylation or dephosphorylation of PDH complex caused by aninhibitor or activator of PDH can be determined by utilizing isoelectricfocusing of PDH complex, for example on a 2-D gel, to cause separationof PDH complex according to the degree of phosphorylation thereof, andvisualizing the separation using a detectably labeled antibody thatbinds specifically to PDH complex in combination with visualization ofthe complex, wherein phosphorylation is indicated by an isoelectricpoint shift associated with a greater negative charge on the complex anddephosphorylation is indicated by an isoelectric point shift associatedwith a less negative charge on the PDH complex as compared with anunphorphorylated PDH complex standard.

In another embodiment, the invention provides a PDH functionalimmunocapture assay for determining interactions between humanmitochondrial PDH complex and known inhibitors, such as sodium arsenite,or activators, such as dichloroacetate, which can be added or removedfrom the captured enzyme in a dose-dependent manner.

In yet another embodiment, the invention provides methods for screeningto detect small molecules, drugs, or proteins that modify the inhibitoror activator activity of a known inhibitor or activator of PDH, forexample by binding to the known compound so as to prevent its inhibitoror activator activity. Such small molecules, drugs, or proteins aredesirable therapeutic agents that could be used to regulate activity ofthe PDH and thereby the energy balance and efficiency of energyutilization of cells and tissues. Such modulators have utility intreatment of disorders of energy production or utilization. Inventioninhibitor screening assay comprises contacting a sample containing PDHcomplex in the presence of a known inhibitor and a test compound with asolid support having attached a PDH complex immunoprecipitating antibodyand determining the degree to which the test compound modifies, i.e.,the inhibits or activates activity of PDH complex the sample.

If combined with quantitation of captured antigen protein, the inventionassays can be used to determine the specific activity (units of activityper mg enzyme protein) of a PDH complex. By this method, a distinctioncan be made between defects in enzyme turnover rates and defects inproduction of sufficient amounts of enzyme.

In still another embodiment, the invention provides isolated monoclonalantibodies characterized as specifically binding to mitochondrial PDHcomplex and immunoprecipitating the entire subunit complex, wherein thecomplex retains functional activity. Also included is a hybridoma cellline that produces a monoclonal antibody having the specificity of amonoclonal antibody as described herein.

The invention also provides a kit for determining PDH activity in a cellcomprising an anti-PDH complex antibody. The kit may contain adetectable label for the antibody, such as a fluorescent label or anenzymatic label.

The assay described herein is based on the specificity of the monoclonalantibodies as well as PDH function. Any general biochemical activity ofPDH function will suffice as a marker of antigen function as theantibody/antigen capture provides specificity to the assay.

In another embodiment, the invention PDH complex functionalimmunocapture assay is suitable for use as a diagnostic assay to detectany type of activity-affecting defect of mitochondrial PDH complex inhumans, such as enzymatic defects, defects in assembly of the enzymecomplex, and the like.

Therefore, the functionality of endogenous PDH complex can be determinedor monitored within small samples, e.g. nanosamples. Such an assay isvaluable, for example, as a research tool for studying the interactionsbetween human mitochondrial PDH complex and its inhibitors andactivators.

In yet another embodiment, the invention provides methods for screeningto detect agents, such as small molecules, drugs, or proteins thatmodify the inhibitor or activator activity of a human mitochondrial PDHcomplex inhibitor or activator, for example by binding to an inhibitorso as to prevent its inhibitor activity. Such small molecules, drugs, orproteins are desirable therapeutic agents that could be used to regulateactivity of PDH complex and thereby the energy balance and efficiency ofenergy utilization of cells and tissues. Such modulators have utility intreatment of disorders of energy production or utilization. Inventionscreening assays comprise contacting a sample containing PDH complex inthe presence of an inhibitor or activator of PDH complex activity and atest active agent and determining the degree to which the test activeagent modifies the inhibitor or activator activity of the PDH complexinhibitor or activator in the sample, wherein a decrease of inhibitoractivity indicates the test active agent inhibits the activity of thePDH complex inhibitor. Active agents that increase activity of a PDHcomplex inhibitor or activator may also be useful in treating disordersof energy production or utilization. The invention screening assay canalso be used to determine the degree to which a test active agentincreases PDH complex inhibitor activity.

If combined with quantitation of captured antigen protein, the inventionassays can be used to determine the specific activity (units of activityper mg enzyme protein) of PDH complex By this method, a distinction canbe made between defects in enzyme turnover rates and defects inproduction of sufficient amounts of enzyme.

In still another embodiment, the invention provides isolated monoclonalantibodies characterized as specifically binding to mitochondrial PDHcomplex and immunoprecipitating the entire complex, wherein the complexretains functional activity. It should be understood that the monoclonalantibody may be able to immunoprecipitate the functional complex in theabsence of all subunits being present, although the presence of allsubunits is preferred.

Also included is a hybridoma cell line that produces a monoclonalantibody having the specificity of a monoclonal antibody as describedherein.

The invention also provides a kit for determining PDH complex activityin a cell comprising an antibody of the invention. The kit may contain adetectable label such as a fluorescent label or an enzymatic label.

The assay described herein is based on the specificity of the monoclonalantibodies as well as antigen function. Any general biochemical activityof antigen will suffice as a marker of antigen function as theantibody/antigen capture provides specificity to the assay.

Alterations in PDH complex reduce or eliminate energy production inmitochondria and so are pathogenic. The literature shows that PDHcomplex is affected by a variety of environmental toxins includingpesticides, impurities in narcotic drugs, and damage to organellescaused by drugs used to treat other diseases, among others. In effectthese inhibit PDH complex activity, for example, enzymatic activity, tovarying degrees. Mutations of PDH complex in patients (geneticallyderived) can also affect activity and produce the same sequellae as thetoxins. Such mutations first affect and then destroy (by apoptosis)those cells with the highest need for ATP. These cells include selectedbrain cells such as those of the substancia nigra cells, whoseimpairment results in Parkinson's disease; frontal cortex cells, whoseimpairment is implicated in Alzheimer's disease or dementia, pancreaticcells, which are involved in insulin secretion; cardiocyte cells, whosedestruction leads to cardiomyopathy, and the like. The present inventionprovides evidence of the utility of antibody analysis in thecharacterization of PDH complex deficiencies of all types.

Accordingly, in one embodiment, the invention provides methods fordetermining the amount of PDH complex in a biological sample of amammalian patient. In this embodiment, the invention assay comprisescontacting isolated antibodies that immunoprecipitate PDH complex with asample comprising solubilized PDH complex so that the antibodies bind toPDH complex present in the sample to form an antibody/PDH compleximmunocomplex, (i.e., under suitable conditions and for a time suitableto form the antibody/PDH complex immunocomplex). Remaining sample, forexample unbound sample contents, is then separated from theimmunocomplex; and the amount of PDH complex in the sample is detected.

Any suitable immunoassay format known in the art and as described hereincan be used to detect and quantify the amount of antibody that binds toan antigen of interest. If the activity of the F1/F0 ATPase in thesample is also known, for example the enzymatic activity, the results ofthe invention method can be used to calculate the specific activity ofthe F1/F0 ATPase in the sample.

As used herein the term “activity” or “functional activity” as appliedto F1/F0 ATPase means all aspects of natural PDH complex activity atphysiological pH, including, but not limited to PDH complex enzymaticactivity in oxidative phosphorylation.

In addition to antibodies that bind to fully assembled PDH complex,antibodies that are known to bind specifically to a particular PDHcomplex subunit can be used to determine the amount of PDH complex in asample, if the antibody immunoprecipitates the full PDH complex. Forexample, an antibody that binds specifically to E-2 subunit can be usedin the invention methods. Although any type of anti-PDH complexantibody, as described herein, that binds specifically to PDH complex orto an identified subunit thereof can be used in the invention methods,monoclonal antibodies are preferred.

Such assays can also be used to determine whether PDH complex isproduced in low quantity as compared with what would be expected in acomparable sample obtained from a normal patient or a normal sample(i.e., obtained from a single patient that has been screened toeliminate the possibility of genetic defects in nucleotide sequencesknown to produce the various peptides that assemble into the PDH complexor from a representative group of such normal patients). For example,“corresponding samples” would be mitochondria isolated from a patientfibroblast cell line and mitochondria isolated from a control skinfibroblast cell line (i.e. isolated from skin fibroblasts of a normalindividual). In addition to fibroblasts, PDH complex-containing samplesfor use in the invention methods can be obtained from whole cellextracts of the patient or from mitochondria isolated from such cells.Although fibroblast cells are particularly convenient as a source ofpatient samples for diagnostic assays, it should be understood that PDHcomplex could be isolated from any mammalian cell, including humancells, with cells having high-energy requirements having the largestsupply of mitochondrial PDH complex. For example, cells that can be usedin the invention methods include neural cells, cardiomyocytes,pancreatic islet cells, hematopoietic cells, liver cells, kidney cells,T cells, B cells and other cell types. Examples of tissue samples thatcan be utilized to obtain cells for use in the invention methods includesaliva, mucosal cells and semen, for example. Alternatively, the assaycan be performed utilizing PDH complex or mitochondria that have beenimmunopurified from patient cells or experimental cells by any methodknown in the art, such as the methods described in the Examples herein.

PDH complex enzymatic activity for energy production in a cell primarilydepends upon the amount of functioning fully assembled PDH complex thatis present in the cell. Therefore, the invention assay can be used todetect a decrease in PHD complex enzymatic activity in the cells of thepatient whose sample is tested, as compared with that of a comparablenormal sample.

The invention immunological tests for PDH complex activity can be usedin a high throughput format using any technique known in the art, suchas FASC screening as is described below in greater detail.

Detectable labels suitable for binding to antibodies used in theinvention methods, including high throughput screening formats, includeradiolabels linked to the antibodies using various chemical linkinggroups or bifunctional peptide linkers. A terminal hydroxyl can beesterified with inorganic acids, e.g., ³²P phosphate, or ¹⁴C organicacids, or else esterified to provide linking groups to the label.Enzymes of interest as detectable labels will primarily be hydrolases,particularly esterases and glycosidases, or oxidoreductases,particularly peroxidases. Fluorescent compounds include fluorescein andits derivatives, rhodamine and its derivatives, dansyl, umbelliferone,and so forth. Chemiluminescers include luciferin, and2,3-dihydrophthalazinediones (e.g., luminol), and the like.

Antibodies may also be attached to solid supports, which areparticularly useful for immunoassays or immunoprecipitation of PDHcomplex. Such solid supports include, but are not limited to, glass,cellulose, polyacrylamide, nylon, polystyrene, polyvinyl chloride orpolypropylene, for example protein G covered wells of microtiter platesor beads.

Antibodies directed against a specific epitope, or combination ofepitopes, so as to bind specifically with the PDH complex will allow forthe screening of patient samples as described herein. Various screeningtechniques can be utilized using such monoclonal antibodies, and includemagnetic separation using antibody-coated magnetic beads, “panning” withantibody attached to a solid matrix (i.e., plate), and flow cytometry(See, e.g., U.S. Pat. No. 5,985,660; and Morrison et al., Cell,96:737-49 (1999)).

The antibodies of the invention may be assayed for immunospecificbinding by any method known in the art. The immunoassays which can beused, include but are not limited to, competitive and non-competitiveassay systems using techniques such as western blots, radioimmunoassays,ELISA (enzyme linked immunosorbent assay), “sandwich” immunoassays,immunoprecipitation assays, precipitin reactions, gel diffusionprecipitin reactions, immunodiffusion assays, agglutination assays,complement-fixation assays, immunoradiometric assays, fluorescentimmunoassays, protein A immunoassays, to name but a few. Such assays areroutine and well known in the art (see, e.g., Ausubel et al, eds, 1994,Current Protocols in Molecular Biology, Vol. 1, John Wiley & Sons, Inc.,New York, which is incorporated by reference herein in its entirety).Exemplary immunoassays are described briefly below (but are not intendedby way of limitation).

Immunoprecipitation protocols generally comprise lysing a population ofcells in a lysis buffer such as RIPA buffer (1% NP-40 or Triton X-100,1% sodium deoxycholate, 0.1% SDS, 0.15 M NaCl, 0.01 M sodium phosphateat pH 7.2, 1% Trasylol) supplemented with protein phosphatase and/orprotease inhibitors (e.g., EDTA, PMSF, aprotinin, sodium vanadate),adding the antibody of interest to the cell lysate, incubating for aperiod of time (e.g., 14 hours) at 4° C., adding protein A and/orprotein G sepharose beads to the cell lysate, incubating for about anhour or more at 4° C., washing the beads in lysis buffer andresuspending the beads in SDS/sample buffer. The ability of the antibodyof interest to immunoprecipitate a particular antigen can be assessedby, e.g., Western blot analysis. Those of skill in the art would beknowledgeable as to the parameters that can be modified to increase thebinding of the antibody to an antigen and decrease the background (e.g.,pre-clearing the cell lysate with sepharose beads). For furtherdiscussion regarding immunoprecipitation protocols see, e.g., Ausubel etal, eds, 1994, Current Protocols in Molecular Biology, Vol. 1, JohnWiley & Sons, Inc., New York at 10.16.1.

Western blot analysis generally comprises preparing protein samples,electrophoresis of the protein samples in a polyacrylamide gel (e.g.,8%-20% SDS-PAGE depending on the molecular weight of the antigen),transferring the protein sample from the polyacrylamide gel to amembrane such as nitrocellulose, PVDF or nylon, blocking the membrane inblocking solution (e.g., PBS with 3% BSA or non-fat milk), washing themembrane in washing buffer (e.g., PBS-Tween 20), blocking the membranewith primary antibody (the antibody of interest) diluted in blockingbuffer, washing the membrane in washing buffer, blocking the membranewith a secondary antibody (which recognizes the primary antibody, e.g.,an anti-human antibody) conjugated to an enzymatic substrate (e.g.,horseradish peroxidase or alkaline phosphatase) or radioactive molecule(e.g., ³²P or 125 I) diluted in blocking buffer, washing the membrane inwash buffer, and detecting the presence of the antigen. Those of skillin the art would be knowledgeable as to the parameters that can bemodified to increase the signal detected and to reduce the backgroundnoise. For further discussion regarding western blot protocols see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 10.8.1.

ELISAs comprise preparing antigen, coating the well of a 96 wellmicrotiter plate with the antigen, adding the antibody of interestconjugated to a detectable compound such as an enzymatic substrate(e.g., horseradish peroxidase or alkaline phosphatase) to the well andincubating for a period of time, and detecting the presence of theantigen. In ELISAs the antibody of interest does not have to beconjugated to a detectable compound; instead, a second antibody (whichrecognizes the antibody of interest) conjugated to a detectable compoundmay be added to the well. Further, instead of coating the well with theantigen, the antibody may be coated to the well. In this case, a secondantibody conjugated to a detectable compound may be added following theaddition of the antigen of interest to the coated well. Those of skillin the art would be knowledgeable as to the parameters that can bemodified to increase the signal detected as well as other variations ofELISAs known in the art. For further discussion regarding ELISAs see,e.g., Ausubel et al, eds, 1994, Current Protocols in Molecular Biology,Vol. 1, John Wiley & Sons, Inc., New York at 11.2.1.

The binding affinity of an antibody to an antigen and the off-rate of anantibody-antigen interaction can be determined by competitive bindingassays. One example of a competitive binding assay is a radioimmunoassaycomprising the incubation of labeled antigen (e.g., ³H or ¹²⁵I) with theantibody of interest in the presence of increasing amounts of unlabeledantigen, and the detection of the antibody bound to the labeled antigen.The affinity of the antibody of interest for a particular antigen andthe binding off-rates can be determined from the data by scatchard plotanalysis. Competition with a second antibody can also be determinedusing radioimmunoassays. In this case, the antigen is incubated withantibody of interest conjugated to a labeled compound (e.g., ³H or ¹²⁵I)in the presence of increasing amounts of an unlabeled second antibody.

Antibodies used in invention assay(s) can be polyclonal, monoclonal, ora functionally active fragment thereof. Mono- or poly-clonal antibodiesto a PDH isoenzyme, its salts, and PDH isoenzyme derivatives, are raisedin appropriate host animals by immunization with invention immunogenicconjugate(s) using conventional techniques as are known in the art.

The preparation of monoclonal antibodies is disclosed, for example, byKohler and Milstein, Nature 256:495-7, 1975; and Harlow et al., in:Antibodies: a Laboratory Manual, page 726 (Cold Spring Harbor Pub.,1988), which are hereby incorporated by reference. Briefly, monoclonalantibodies can be obtained by injecting mice, or other small mammals,such as rabbits, with a composition comprising an invention immunogenicconjugate whose preparation is disclosed above, verifying the presenceof antibody production by removing a serum sample, removing the spleento obtain B lymphocytes, fusing the B lymphocytes with myeloma cells toproduce hybridomas, cloning the hybridomas, selecting positive clonesthat produce antibodies to the antigen, and isolating the antibodiesfrom the hybridoma cultures. Monoclonal antibodies can be isolated andpurified from hybridoma cultures by a variety of well-establishedtechniques. Such isolation techniques include affinity chromatographywith Protein-A Sepharose, size-exclusion chromatography, andion-exchange chromatography. See, for example, Barnes et al.,Purification of Immunoglobulin G (IgG), in: Methods in Mol. Biol., 10:79-104, 1992). Antibodies of the present invention may also be derivedfrom subhuman primate antibodies. General techniques for raisingantibodies in baboons can be found, for example, in Goldenberg et al.,International Patent Publication WO 91/11465 (1991) and Losman et al.,Int. J. Cancer, 46:310-314, 1990.

It is also possible to use anti-idiotype technology to producemonoclonal antibodies which mimic an epitope. For example, ananti-idiotypic monoclonal antibody made to a first monoclonal antibodywill have a binding domain in the hypervariable region which is the“image” of the epitope bound by the first monoclonal antibody.

The term “antibody” as used in this invention includes intact moleculesas well as functional fragments thereof, such as Fab, F(ab′)₂, and Fvthat are capable of binding a PDH isoenzyme, or a salt thereof,especially after the PDH isoenzyme or salt thereof has been derivatizedwith a linker molecule as disclosed herein. These functional antibodyfragments are defined as follows:

-   -   (1) Fab, the fragment which contains a monovalent        antigen-binding fragment of an antibody molecule, can be        produced by digestion of whole antibody with the enzyme papain        to yield an intact light chain and a portion of one heavy chain;    -   (2) Fab′, the fragment of an antibody molecule that can be        obtained by treating whole antibody with pepsin, followed by        reduction, to yield an intact light chain and a portion of the        heavy chain; two Fab′ fragments are obtained per antibody        molecule;    -   (3) (Fab′)₂, the fragment of the antibody that can be obtained        by treating whole antibody with the enzyme pepsin without        subsequent reduction; F(ab′)₂ is a dimer of two Fab′ fragments        held together by two disulfide bonds;    -   (4) Fv, defined as a genetically engineered fragment containing        the variable region of the light chain and the variable region        of the heavy chain expressed as two chains; and    -   (5) Single chain antibody (“SCA”), a genetically engineered        molecule containing the variable region of the light chain and        the variable region of the heavy chain, linked by a suitable        polypeptide linker as a genetically fused single chain molecule.

Methods of making these fragments are known in the art. (See forexample, Harlow and Lane, Antibodies: A Laboratory Manual, Cold SpringHarbor Laboratory, New York, 1988, incorporated herein by reference). Asused in this invention, the term “epitope” means any antigenicdeterminant on an antigen to which the paratope of an antibody binds.Epitopic determinants usually consist of chemically active surfacegroupings of molecules such as amino acids or carbohydrate side chainsand usually have specific three dimensional structural characteristics,as well as specific charge characteristics.

Antibody fragments according to the present invention can be prepared byproteolytic hydrolysis of the antibody or by expression in E. coli ofDNA encoding the fragment. Antibody fragments can be obtained by pepsinor papain digestion of whole antibodies by conventional methods. Forexample, antibody fragments can be produced by enzymatic cleavage ofantibodies with pepsin to provide a 5S fragment denoted F(ab′)₂. Thisfragment can be further cleaved using a thiol reducing agent, andoptionally a blocking group for the sulfhydryl groups resulting fromcleavage of disulfide linkages, to produce 3.5S Fab′ monovalentfragments. Alternatively, an enzymatic cleavage using pepsin producestwo monovalent Fab′ fragments and an Fe fragment directly. These methodsare described, for example, by Goldenberg, U.S. Pat. Nos. 4,036,945 and4,331,647, and references contained therein, which patents are herebyincorporated by reference in their entirety. See also Porter, R. R.,Biochem. J., 73: 119-126, 1959. Other methods of cleaving antibodies,such as separation of heavy chains to form monovalent light-heavy chainfragments, further cleavage of fragments, or other enzymatic, chemical,or genetic techniques may also be used, so long as the fragments bind tothe antigen that is recognized by the intact antibody.

Fv fragments comprise an association of V_(H) and V_(L) chains. Thisassociation may be noncovalent, as described in Inbar et al., Proc.Nat'l Acad. Sci. USA 69:2659-62, 1972. Alternatively, the variablechains can be linked by an intermolecular disulfide bond or cross-linkedby chemicals such as glutaraldehyde. Preferably, the Fv fragmentscomprise V_(H) and V_(L) chains connected by a peptide linker. Thesesingle-chain antigen binding proteins (sFv) are prepared by constructinga structural gene comprising DNA sequences encoding the V_(H) and V_(L)domains connected by an oligonucleotide. The structural gene is insertedinto an expression vector, which is subsequently introduced into a hostcell such as E. coli. The recombinant host cells synthesize a singlepolypeptide chain with a linker peptide bridging the two V domains.Methods for producing sFvs are described, for example, by Whitlow andFilpula, Methods, 2: 97-105, 1991; Bird et al., Science 242:423-426,1988; Pack et al., Bio/Technology 11:1271-77, 1993; and Ladner et al.,U.S. Pat. No. 4,946,778, which is hereby incorporated by reference inits entirety.

Another form of an antibody fragment is a peptide coding for a singlecomplementarity-determining region (CDR). CDR peptides (“minimalrecognition units”) can be obtained by constructing genes encoding theCDR of an antibody of interest. Such genes are prepared, for example, byusing the polymerase chain reaction to synthesize the variable regionfrom RNA of antibody-producing cells. See, for example, Larrick and Fry,Methods, 2: 106-10, 1991.

The invention methods use monoclonal antibodies characterized asspecifically binding to PDH complex of the mitochondrial respiratorychain and immunoprecipitating PDH complex, wherein PDH complex retainsfunctional activity.

Hybridoma cell lines producing monoclonal antibodies useful in theinvention methods for immunocapture of PDH complex (while allowing thePDH complex to remain enzymatically active) are commercially availableby hybridoma name as used to identify the monoclonal antibodies fromMolecular Probes (Eugene, Oreg.) or from the Monoclonal AntibodyFacility at the University of Oregon (Eugene, Oreg.).

The invention is further illustrated by the following non-limitingexamples:

EXAMPLE 1

Materials and Methods

Monoclonal Antibodies

Monoclonal antibodies (“MAbs) used in this study were developed in theUniversity of Oregon Monoclonal Antibody Facility, Eugene, Oreg.Anti-subunit and E2IE3 bp subunit mAbs were generated respectively byimmunizing mice with purified porcine PDH complex [26]. Antibodies werescreened first for binding to purified porcine PDH (Sigma) and then forspecific binding to a single subunit in denaturing Western blots of bothpure porcine PDH and human mitochondria.

Preparation of Heart Mitochondria

Bovine heart mitochondria were prepared as described in Hanson et al.without modifications [27]. Human heart tissue was provided andmitochondria prepared by Analytic Biological Services, Inc. according toSmith [28].

Lysis of Mitochondria

The entire procedure was performed at 4° C. in the presence ofproteinase inhibitors (leupeptin 0.5 μg/ml, pepstatin 0.5 μg/ml and 1 mMPMSF). Mitochondria were diluted with PBS buffer (100 mM NaCl, 80 mMNaH₂PO₄, 20 mM NaH₂PO₄ pH 7.5) to a total concentration of 4 mg/ml inEppendorf tubes and lysed in the presence of 0.4% lauryl maltoside.After 20 mm incubation on ice with occasional mixing, tubes werecentrifuged at 14,000 rpm at 4° C. for 10 mm in an Eppendorf microfuge.The supernatant was collected and protein concentration was determinedby Bradford assay. The supernatant was immediately used forimmunoprecipitation and activity studies. For immunoprecipitation, thesupernatant fraction was precleared by incubation of the lysedmitochondria with protein G agarose beads at 4° C. for 2 hours tominimize non-specific binding.

Cell Preparation

Normal human fibroblasts (MRC5) were obtained from the American TypeCulture Collection (Manassas, Va.). Patients fibroblasts, obtained fromskin biopsies of patients, were kindly provided by Dr. Nancy Kennaway,Oregon Health Sciences University, after patient consent. These includedconfirmed (TC404, TC581) or suspected (TC594) defects in expression ofthe E1α subunit. Cells were grown in high-glucose Dulbecco's modifiedEagle's medium supplemented with 10% fetal calf serum and 50 μg/mluridine. Cells were grown to confluency on 150 mm² plates, washed withcalcium- and magnesium-free PBS (CMF-PBS), and than trypsinized forcollection into 15 ml tubes. Harvested cells were washed with CMF-PBS 3times and frozen at −80° C. for at least one hour. Cells than wereresuspended in 200 μl of CMF-PBS with protein inhibitors (leupeptin 0.5μg/ml, pepstatin 0.5 μg/ml and PMSF mM) and lysed in the presence oflauryl maltoside solution (total concentration 0.5%). After 30 min ofincubation on ice with occasional mixing, cells were centrifuged for 20minutes at 4° C. in Eppendorf microfuge (˜16000 g). Supernatant wassaved, analyzed for protein concentration by Bradford assay andimmediately used for activity measurements or for immunoprecipitationexperiment.

EXAMPLE 2

Immunoprecipitation of MRC5 Fibroblasts Bovine and Human Heart PDHComplex

All procedures were performed in the presence of protein inhibitors inthe concentrations described above. The antibody column was prepared byincubating 25 mg protein G agarose beads with 80 μg/reaction of eitheranti-E2 specific antibody or normal mouse IgG as the negative control atroom temperature for 2 hours. To couple antibodies to the protein Gbeads, the beads were washed twice with 0.2M sodium borate and incubatedwith 20 mm dimethylpimelimidate in 0.2M sodium borate for 30 mm. Then,the beads were washed twice with 0.2 ethanolamine (pH 8) and incubatedin the same solution for another 2 hours to stop the reaction.Afterwards, the beads were washed 3 times with PBS buffer in thepresence of proteinase inhibitors, and precleared supernatant wasapplied. Mitochondria were incubated with the antibody column overnightat 4° C. and the precipitated PDH complexes were washed 5 times with PBSbuffer with protease inhibitors in the presence of 0.02% laurylmaltoside. Protein was released by 20 min incubation with 30-40 μl perreaction of 100 mM glycine, pH 2.5. After a brief centrifugation, thefractions were equilibrated to pH 7.5 with mM Tris-base solution,dissolved in DS-PAGE tricine sample buffer (BioRad) containing 2%β-mercaptoethanol, and the proteins separated on a 10%SDS-polyacrylamide gel at 100 V for ˜20 mm (stacking gel) and then at200 V for ˜40 mm (separating gel) loaded on SDS-PAGE. The gel wasstained by silver-staining procedure as described previously [29].

Determination of PDH Activity

96-well ELISA plates (Falcon Probind) were coated with Goat-anti-mouseIgG Fc specific antibody (0.5 μg/well) in PBS buffer overnight at 4° C.After 3 washings, wells were coated with monoclonal anti-PDH-E2 subunitat 0.5 μg/well and incubated for 1-2 hours at room temperature. Thewells were washed 3-4 times and covered with a 0.5% bovine serum albuminsolution for 1 hour to minimize non-specific protein binding to thewells. After blocking, wells were washed 5 times and 100 μl ofsolubilized mitochondria applied and incubated in the wells at roomtemperature for 1 hour. After incubation, the supernatant was collectedand reapplied to a second antibody-covered plate to test for residualactivity.

100 μl/well of the reaction mixture (50 mM Tris, pH 7.5; 2 mM β-NAD+,225 μM TPP, 2 mM pyruvate, 150 μM Coenzyme A, 2.6 mM cysteine, 1 mMMgCl₂) was applied and NADH production was monitored eitherspectrophotometrically at 340 nM or by measuring fluorescence in thepresence of 15 μM resazurin and 0.5 U/ml diaphorase using an excitationwavelength of 530 nm and an emission wavelength of 590 nm for up to 75mm. Different concentrations of NADH were loaded in the presence ofreaction mixture to create a standard curve on each plate. Activity wascalculated as nMol NADH/min/mg of mitochondrial protein.

Results

Human Heart Bovine Heart and MRC5 Fibroblasts' Pyruvate DehydrogenaseComplexes can be Immunoprecipitated with an Anti-E2 mAb

Monoclonal antibodies raised against porcine pyruvate dehydrogenasecomplex were found to cross react with both human and bovine PDH onWestern blots and in immunocytochemistry. One of the antibodies specificto the E2 subunit was found to immunoprecipitate the PDH complex fromdetergent solubilized human fibroblasts, human heart mitochondria andbovine heart mitochondria. The polypeptide profile of theimmunoprecipitate was similar to that of conventionally purifiedmammalian PDH [30, 31] and shows 5 major bands, corresponding tosubunits E2 (72 kDa), E3 (55 kDa), E3bp (50 kDa), E₁α (41 kDa) and E₁β(36 kDa), as confirmed by peptide sequencing. There was no evidence ofco-precipitation of the 2-oxo-acid dehydrogenase from the gel profilesor from sequencing of bands to ensure that subunits of this enzyme werenot co-migrating with PDH subunits. The immunoprecipitated complexproved to be active when bound to antibody on the beads, based onproduction of NADH in the presence of the required substrates and, asshown below, the immunocapture method can be adapted to measure PDHcomplex activity on microplates, for example, 96-well microplates.

The Activity of Immunocaptured PDH is Substrate and Co-Factor Dependent

Monoclonal anti-E2 antibodies were attached to 96-well microplates towhich had been prebound goat anti-mouse IgG Fc mAbs, as described inMaterials and Methods. After appropriate blocking and washing,detergent-solubilized mitochondria, or cell extracts in the case offibroblasts, were applied to the wells and incubated at room temperaturefor at least an hour. Unbound protein was removed by extensive washingsand NADH production then measured by the increase of absorbance at 340nM or by coupled fluorescence assay, in the presence of NAD+, pyruvate,CoA, TPP, MgCl₂, and cysteine as a reducing agent in 50 mM Tris-HClbuffer pH 7.8. FIG. 1 shows the activity of immunocaptured human heartPDH measured at 37° C. for an hour. When one of the substrates of thereaction was omitted, the absorbance remained at the background levelwith the exception of when TPP was omitted, where 30% total PDH activitywas detected, suggesting that this co-factor is co-immunoprecipitatedwith the enzyme but in less than stoichiometric amounts, with respect tothe E1 subunits. The same results were obtained with the fluorescentassay. As expected, the total PDH activity captured in each well of themicroplate ate depends on the amount of capture mAb bound. At a fixedconcentration of antibody (0.5 μg/well), there was a good linearcorrelation of activity with protein concentration from 6 μg to 50μg/well (spectrophotometric measurements) or from 0.4 μg to 50 μg/well(fluorescence measurements) for both human and bovine heart mitochondriapreparations.

The amount of protein bound to the mAb per well was determined asfollows: increasing amounts of mitochondrial protein were added to wellsand allowed to bind to the capture MAb. Supernatants from each well werethen transferred to a second plate coated with the same mAb. Theactivity captured by the second plate then measures enzyme levels inexcess of what are needed to saturate the first plate (FIG. 2). Atconcentrations of 50 μg of mitochondrial protein, all of the activitywas bound up by the first plate, but above this there was unbound enzymefor capture on the second plate. Based on this result, it can be assumedthat when samples of above 50 μg are used (and excess is washed awaybefore the assay), the activity being measured is for 50 μg protein.Thus, as shown in FIG. 2, for human heart, the PDH activity was5.50±0.33 U, where a unit is 1 μmol of NADH produced per min/mgmitochondrial protein. The value obtained for bovine heart was 6.61±1.0U, and these values are within the range of previously publishedactivities for an enzyme in various purified mitochondrial preparationsor tissues [32-36].

PDH can be Inhibited by Sodium Arsenite, ATP and Anti-Lipoic AcidMonoclonal Antibody

The functionality of PDH bound to the microtiter plates was furtherexamined in the presence of several different known inhibitors andactivators of the enzyme complex. Immunocaptured PDH was inhibited bysodium arsenite by >98%. In the presence of ATP, PDH activity wasinhibited by about 70%. Dichloroacetate increased the activity of theenzyme by 34%. FIG. 3 summarizes the results of these studies ofinhibition and activation of PDH activity. Recently, an anti-E2/E3 bpantibody has been identified as being specific for the lipoylated formof the lipoyl domain on both of these subunits. As shown, this mAbinhibited PDH activity to the background level. Thus the anti-E2/E3antibody can be used in treatments where it is desirable to reduce PDHactivity.

A Shift in Isoelectric Focusing Point can be Used to Determine theDegree of Phosphorylation of PDH

The ability of ATP to inhibit PDH suggested that the enzyme was beingimmunocaptured with a significant amount of PDH kinase bound. To testthis further, samples were treated with ATP or dichloroactetate (DCA)and then subjected to 2D-gel electrophoresis. Previous studies haveestablished that differently-phosphorylated forms of the E1α subunit canbe resolved by the isoelectric focusing step, wherein the molecules areseparated on a pH gradient by the degree of phosphorylation becauseaddition of P_(i) to the protein adds negative charge and shifts thespot of a phosphorylated subunit upon isoelectric focusing. [37].Isoelectric focusing studies were conducted using a pH gradient from pH10 to pH 3 to follow this shift for small amounts of PDH by using theE1α-specific mAb in Western blots of the 2D gels. As evident in FIG. 3,ATP reaction greatly increased the level of phosphorylation while DCA,which inhibits PDH kinases and therefore increases dephosphorylation,reduced it to below that of untreated enzyme.

Assay of PDH Activity in Patients with PDH Deficiency

FIG. 4 shows the immunocapture assay used to detect PDH deficiency inpatients. In these experiments, PDH was immunocaptured on the microtiterplates from cell lysates of human fibroblasts (MRC5) to minimize theamount of protein needed and from fibroblast cultures from 3 differentpatients, each with a mutation in the E1α gene. Pyruvate-dependent NADHproduction was followed fluorometrically using resazurin and diaphoraseat an excitation wavelength of 530 um and an emission wavelength of 590nm. The activity of the PDH in deficient fibroblasts was calculated as apercentage relative to that in normal fibroblasts (MRC5). The percent ofnormal PDH activity for TC404, TC594 and TC58 1 fibroblast cell lineswas measured to be 7-10%, 65-78%, and 14-18% respectively, valuesconsistent with the earlier reports of PDH activities obtained for thesecell lines using the [¹⁴C] pyruvate-based activity assay [38]. Theresults of this study are summarized in Table 1 below: TABLE 1Activities of the pyruvate dehydrogenase complex in patient fibroblastsPDH deficient cell PDH activity estimated in Previously reported linesthis study PDH activity [38] TC404 7-10% 16-28% in lympho- cytes TC58114.2-20% 10-17% in fibroblasts TC594 64.2%-78% 37-41% in fibroblastsreferred to as “Just be- low the control range”

In combination with isoelectric focusing, the invention methods can alsobe used to determine the degree of phosphorylation of PDH, a mechanismthat regulates glucose metabolism.

Discussion

Thus, the invention provides methods to measure PDH activity in crudemitochondrial extracts. This method is based on the ability of amonoclonal anti-E2 PDH subunit antibody to immunoprecipitate (capture)fully assembled active PDH complex from detergent-solubilized human andbovine heart mitochondrial extracts. This assay is readily adapted foruse on microtiter plates, such as 96-well plates. The production of NADHin the presence of enzyme substrates can also be monitoredspectrophotometrically or fluorometrically after the enzyme is capturedby immobilized antibody on the well and all interfering enzymeactivities were washed away. A number of methods already exist formeasuring PDH activity. The most commonly used in clinical practicemonitors the formation of [¹⁴C] CO₂ from radioactive pyruvate or lactate[39]. However, the use of radioactivity is difficult and limited to veryfew clinical centers, and is particularly problematic whenhigh-throughput is needed. Another method described recently monitorsacetyl CoA production by its reaction with a dye, acetyl CoAacrylamine-N-acetyl transferase [40]. This method requires the elaboratepurification of the acrylamine-acetyltransferase from pigeon heart.Moreover, both methods measure the utilization of substrate orproduction formation, which for this enzyme are components metabolizedby many other enzymes of the cell. Hence, many different controls areneeded that are not routinely used. The invention methods provide asignificant advantage in that PDH can be purified away from both otherpyruvate metabolizing enzymes, as well as the many NADH-producing andNADH-utilizing enzymes in cells, which otherwise would considerablycomplicate PDH activity measurements.

Using the invention methods, the immunocaptured PDH complex enzyme hasinhibited by previously described PDH inhibitors such as sodium arseniteand ATP [41, 42]. Also, immunoprecipitated PDH complex can be activatedby DCA, a previously described PDH complex activator. [43-45]. ATP orDCA was added to immunoprecipitated bovine heart PDH and theimmunocomplex was resolved on two-dimensional gel by electrophoresisover a pH range from 10 to 3. The results of this study showed thatknown PDH complex inhibitor ATP, not only inhibits enzyme turnover, butalso increases the phosphorylation patterns of E1α. This 2-D gelelectrophoresis data for anti-PDH immunoprecipitated human and bovineheart PDH complex provide evidence for the presence of both PDH kinasesand PDH phosphatases in the enzyme complex as isolated using theinvention methods.

Thus, the invention methods provide a new assay procedure thatsimplifies the diagnosis of PDH deficiencies. Heretofore, activitydefects of patients have been analyzed at only a few large clinicalcenters set up for reproducible assay of the enzyme using [¹⁴C] pyruvateand measuring [¹⁴C] CO₂ production. The invention assays describedherein are much more user friendly and provide a high throughputprocedure for examining samples from multiple patients and controls atonce.

The invention immunocapture method is useful in studies of the severaldiseases where PDH activity changes occur, e.g., diabetes [3]. Moreover,the ability to isolate the enzyme so that endogenous phosphorylationlevels can be determined opens up several new avenues of research.

REFERENCES

-   1. Cross, J. H., Connelly, A., Gadian, D. G., Kendall, B. E.,    Brown, G. K., Brown, R. M., and Leonard, J. V. (1994) Clinical    diversity of pyruvate dehydrogenase deficiency. Pediair. Neurol. 10,    276-283.-   2. Patel, M. S., and Harris, R. A. (1995) Mammalian alpha-keto acid    dehydrogenase complexes: gene regulation and genetic defects.    Faseb J. 9, 1164-1172.-   3. Wu, P., Sato, I., Zhao, Y., Jaskiewicz, J., Popov, K. M., and    Harris, R. A. (1998) Starvation and diabetes increase the amount of    pyruvate dehydrogenase kinase isoenzyme 4 in rat heart. Biochem. J.    329 (Pt 1), 197-201.-   4. Bigl, M., Bruckner, M. K., Arendt, T., Bigl, V., and    Eschrich, K. (1999) Activities of key glycolytic enzymes in the    brains of patients with Alzheimer's disease. J. Neural. Transm. 106,    499-511.-   5. Vary, T. C., and Hazen, 5. (1999) Sepsis alters pyruvate    dehydrogenase kinase activity in skeletal muscle. Mol. Cell.    Biochem. 198, 113-118.-   6. Heininger, K (2000) A unifying hypothesis of Alzheimer's    disease. IV. Causation and sequence of events. Rev. Neurosci. 11    Spec No, 213-328.-   7. Roche, T. E., Baker, J. C., Yan, X., Hiromasa, Y., Gong, X.,    Peng, T., Dong, I., Turkan, A., and Kasten, S. A. (2001) Distinct    regulatory properties of pyruvate dehydrogenase kinase and    phosphatase isoforms. Prog. Nucleic Acid Res. Mol. Biol. 70, 33-75.-   8. Reed, L. J., Lawson, J. E., Niu, X. D., Yazdi, M. A.,    Fussey, S. P. (1992) Biochemical and molecular genetic aspects of    eukaryotic pyruvate dehydrogenase multienzyme complexes. J. Nutr.    Sci. Vitaminol. (Tokyo) Spec, 46-51.-   9. Jilka, J. M., Rahmatullah, M., Kazemi, M., and    Roche, T. E. (1986) Properties of a newly characterized protein of    the bovine kidney pyruvate dehydrogenase complex. J. Biol. Chem.    261, 1858-1867.-   10. Patel, M. S., and Roche, T. E. (1990) Molecular biology and    biochemistry of pyruvate dehydrogenase complexes. Faseb J. 4,    3224-3233.-   11. Gudi, R., Bowker-Kinley, M M., Kedishvili, N. Y., Zhao, Y., and    Popov, K M. (1995) Diversity of the pyruvate dehydrogenase kinase    gene family in humans. J. Biol. Chem. 270, 28989-28994.-   12. Bowker-Kinley, M. M., Davis, W. I., Wu, P., Harris, R. A., and    Popov, K. M. (1998) Evidence for existence of tissue-specific    regulation of the mammalian pyruvate dehydrogenase complex.    Biochem. J. 329 (Pt 1), 191-196.-   13. Bowker-Kinley, M., and Popov, K. M. (1999) Evidence that    pyruvate dehydrogenase kinase belongs to the ATPase/kinase    superfamily. Biochem. J. 344 Pt 1, 47-53.-   14. Wu, P., Inskeep, K., Bowker-Kinley, M. M., Popov, K. M., and    Harris, R, A, (1999) Mechanism responsible for inactivation of    skeletal muscle pyruvate dehydrogenase complex in starvation and    diabetes. Diabetes 48, 1593-1599.-   15. Baker, J. C., Yan, X., Peng, T., Kasten, S., and    Roche, T. E. (2000) Marked differences between two isoforms of human    pyruvate dehydrogenase kinase. J. Biol. Chem. 275, 15773-15781.-   16. Steussy, C. N., Popov, K. M., Bowker-Kinley, M. M., Sloan, R.    B., Jr., Harris, R. A., and Hamilton, J. A. (2001) Structure of    pyruvate dehydrogenase kinase. Novel folding pattern for a serine    protein kinase. J. Biol. Chem. 276, 37443-37450.-   17. Sugden, P. H., Hutson, N. J., Kerbey, A. L., and    Randle, P. J. (1978) Phosphorylation of additional sites on pyruvate    dehydrogenase inhibits its re-activation by pyruvate dehydrogenase    phosphate phosphatase. Biochem. J. 169, 433-435.-   18. Sugden, P. H., and Randle, P. J. (1978) Regulation of pig heart    pyruvate dehydrogenase by phosphorylation. Studies on the subunit    and phosphorylation stoichiometries. Biochem. J. 173, 659-668.-   19. Sugden, P. H., and Simister, N. E. (1980) Role of multisite    phosphorylation in the regulation of ox kidney pyruvate    dehydrogenase complex. FEBS LetL 111, 299-302.-   20. Korotchkina, L. G., Khailova, L. S., and Severin, S. E. (1995)    The effect of phosphorylation on pyruvate dehydrogenase. FEBS Lett.    364, 185-188.-   21. Chow, C. W., and Thorburn, D. R. (2000) Morphological correlates    of mitochondrial dysfunction in children. Hum. Reprod. 15 Suppl 2,    68-78.-   22. Nissenkorn, A., Michelson, M., Ben-Zeev, B., and    Lerman-Sagie, T. (2001) Inborn errors of metabolism: a cause of    abnormal brain development. Neurology 56, 1265-1272.-   23. De Vivo, D. C. (1998) Complexities of the pyruvate dehydrogenase    complex. Neurology 51, 1247-1249.-   24. Shany, E., Saada, A., Landau, D., Shaag, A., Hershkovitz, E.,    and Elpeleg, O. N. (1999) Lipoamide dehydrogenase deficiency due to    a novel mutation in the interface domain. Biochem. Biophys. Res.    Commun. 262, 163-166.-   25. Brown, R. M., Head, R. A., and Brown, G. K. (2002) Pyruvate    dehydrogenase E3 binding protein deficiency. Hum. Genet. 110,    187-191.-   26. Lib, M. Y., Brown, R. M., Brown, G. K., Marusich, M. F., and    Capaldi, R. A. (2002) Detection of pyruvate dehydrogenase E1    alpha-subunit deficiencies in females by immunohistochemical    demonstration of mosaicism in cultured fibroblasts. J. Histochem.    Cytochem. 50, 877-884.-   27. Hanson, B. J., Schulenberg, B., Patton, W. F., and    Capaldi, R. A. (2001) A novel subfractionation approach for    mitochondrial proteins: a three-dimensional mitochondrial proteome    map. Electrophoresis 22, 950-959.-   28. Smith, S., Cottingham, I. R., and Ragan, C. I. (1980)    Immunological assays of the NADH dehydrogenase content of bovine    heart mitochondria and submitochondrial particles. FEBS Lett 110,    279-282.-   29. Lauber, W. M., Carroll, J. A., Dufield, D. R., Kiesel, J. R.,    Radabaugh, M. R., and Malone. J. P. (2001) Mass spectrometry    compatibility of two-dimensional gel protein stains. Electrophoresis    22, 906-918.-   30. Roche, T. E., and Cate, R. L. (1977) Purification of porcine    liver pyruvate dehydrogenase complex and characterization of its    catalytic and regulatory properties. Arch. Biochem. Biophys. 183,    664-677.-   31. Harris, R. A., Popov, K. M., Shimomura, Y., Zhao, Y.,    Jaskiewicz, J., Nanaumi, N., and Suzuki, M. (1992) Purification,    characterization, regulation and molecular cloning of mitochondrial    protein kinases. Adv. Enzyme Regul. 32, 267-284.-   32. Hinman, L. M., and Blass, J. P. (1981) An NADH-linked    spectrophotometric assay for pyruvate dehydrogenase complex in crude    tissue homogenates. J. Biol. Chem. 256, 6583-6586.-   33. GOHIL AND JONES, 1983-   34. Sheu, K. F., Lai, J. C., Kim, Y. T., Dorante, G., and    Bagg, J. (1985) Immunochemical characterization of pyruvate    dehydrogenase complex in rat brain. J. Neurochem. 44, 593-599.-   35. Scislowski, P. W., and Davis, E. J. (1986) A sensitive    spectrophotometric assay of pyruvate dehydrogenase activity. Anal.    Biochem. 155, 400-404.-   36. Haas, R. H., Thompson, G., Morris, B., Conright, K., and    Andrews, T. (1988) Pyruvate dehydrogenase activity in osmotically    shocked rat brain mitochondria: stimulation by oxaloacetate. J.    Neurochem. 50, 673-680.-   37. Wicking, C. A., Scholem, R. D., Hunt, S. M., Brown, G. K. (1986)    Immunochemical analysis of normal and mutant forms of human pyruvate    dehydrogenase. Biochem. J. 239, 89-96.-   38. Wexler, I. D., Kerr, D. S., Ho, L., Lusk, M. M., Pepin, R. A.    Javed, A. A., Mole, J. E., Jesse, B. W., Thekkurnkara, T. J., Pons,    G., et al. (1988) Heterogeneous expression of protein and mRNA in    pyruvate dehydrogenase deficiency. Proc. Natl. Acad. Sci. USA 85,    7336-7340.-   39. Schofield, P. J., Griffiths, L. R., Rogers, S. H., and    Wise, G. (1980) An improved method for the assay of platelet    pyruvate dehydrogenase. Clin. Chim. Acta 108, 219-227.-   40. Brooks, S. P., and Storey, K. B. (1993) An improvement in the    pyruvate dehydrogenase complex assay: a high-yield method for    purifying arylamine acetyltransferase. Anal. Biochem. 212,452-456.-   41. Walsh, D. A., Cooper, R. H., Denton, R. M., Bridges, B. J., and    Randle, P. J. (1976) The elementary reactions of the pig heart    pyruvate dehydrogenase complex. A study of the inhibition by    phosphorylation. Biochem. J. 157, 41-67.-   42. Petrick, J. S., Jagadish, B., Mash, E. A., and    Aposhian, H. V. (2001) Monomethylarsonous acid (MMA(III)) and    arsenite: LD(50) in hamsters and in vitro inhibition of pyruvate    dehydrogenase. Chem. Res. Toxicol. 14, 651-656.-   43. Evans, O. B. (1982) Dichloroacetate tissue concentrations and    its relationship to hypolactatemia and pyruvate dehydrogenase    activation. Biochem. Pharmacol. 31, 3124-3126.-   44. Evans, O. B. (1983) Effects of dichloroacetate on brain tissue    pyruvate dehydrogenase. J. Neurochem. 41, 1052-1056.-   45. Gibala, M. J., and Saltin, B. (1999) PDH activation by    dichloroacetate reduces TCA cycle intermediates at rest but not    during exercise in humans. Am. J. Physiol. 277, 33-38.

Although the invention has been described with reference to the aboveexamples, it will be understood that modifications and variations areencompassed within the spirit and scope of the invention. Accordingly,the invention is limited only by the following claims.

1. A method for determining the amount of pyruvate dehydrogenase (PDH)complex in a biological sample compromising: contacting a samplecomprising PDH complex with an isolated antibody that specifically bindsto PDH complex under conditions to allow specific binding of theantibody to solubilized PDH complex present in the sample to form animmunocomplex; separating remaining sample contents from theimmunocomplex; and detecting the amount of the PDH complex in theseparated immunocomplex, thereby determining the amount of the PDHcomplex in the biological sample.
 2. The method of claim 1, wherein theantibody is an anti-E2 specific antibody, a monoclonal antibody, or amonoclonal anti-E2 specific antibody. 3-5. (canceled)
 6. The method ofclaim 1, wherein the PDH complex in the immunocomplex retains PDHactivity.
 7. The method of claim 1, wherein the antibody is attached toa solid support and the separating includes separating unbound samplecontents from the solid support.
 8. The method of claim 7, wherein theseparating comprises: (i) releasing the immunocomplex complex; and (ii)separating the immunocomplex from other components of the sample usingSDS-PAGE.
 9. The method of claim 7, wherein the detecting comprisescontacting immunocomplexed PDH complex with a detectable marker thatbinds specifically to the immunocomplexed PDH and measuring the amountof detectable marker present on the solid support.
 10. The method ofclaim 7, wherein the solid support is a microtiter plate or beads.11-12. (canceled)
 13. The method of claim 1 further comprising: (i)quantifying the immunocaptured PDH complex detected in the sample bycomparing with a standard reference curve obtained using a purifiedsample of PDH complex; (ii) determining specific activity of theimmunocaptured PDH complex; or (iii) both (i) and (ii).
 14. (canceled)15. The method of claim 13, wherein the sample is obtained from apatient sample and wherein the method further comprises distinguishingbetween a defect in PDH complex turnover rate and a defect in productionof PDH complex in the patient.
 16. A method for measuring activity ofPDH complex in a sample, said method comprising: a) contacting a samplecomprising PDH complex with an isolated antibody that specifically bindto PDH complex under conditions to allow formation of an immunocomplexof the antibody and the PDH complex present in the sample; b) contactingthe immunocomplex with a reaction mixture comprising a non-limitingamount of one or more substrates necessary for activity of the PDHcomplex; and c) detecting: (i) the amount of NADH produced in thereaction mixture, wherein the amount of NADH produced indicates theactive state of the PDH complex; or (ii) the level of phosphorylation ofimmunocomplexed PDH complex in the in the sample as compared with thatof an unphoshorylated PDH complex standard, wherein a level ofphosphorylation greater than that in the standard indicates a loweredlevel of activity, and a level of phosphorylation substantially equal tothat of the PDH complex in the sample indicates a normal level ofactivity of the PDH complex in the sample.
 17. (canceled)
 18. The methodof claim 16, wherein the substrates are β-NAD⁺, Coenzyme A, FAD⁺,cysteine, pyruvate, and thiamine pyrophosphate (TPP).
 19. The method ofclaim 16, wherein the detecting comprises: (i) transferring an electronfrom reduced NADH to an electron acceptor molecule to produce NADH; and(ii) determining a change indicating transfer of an electron to theelectron acceptor molecule, wherein magnitude of the change indicatesbiological activity of the PDH complex.
 20. The method of claim 19,wherein the electron acceptor molecule is an electron acceptor dyemolecule; and wherein determining a change indicating transfer of anelectron involves monitoring the reaction mixture spectrophotometricallyto detect a change in absorbance of the electron acceptor dye molecule;wherein magnitude of the change indicates biological activity of the PDHcomplex as compared to that of a comparable healthy sample of PDHcomplex.
 21. The method of claim 20, wherein the electron acceptor dyemolecule is selected from diaphorase, resazurin, and a combinationthereof.
 22. The method of claim 20, wherein the monitoring comprisesdetecting a change in fluorescence from the dye molecule.
 23. The methodof claim 20, wherein the detecting comprises: (i) contacting thereaction mixture with a PDH inhibitor and comparing an amount ofresultant inhibition of the PDH complex compared to that of a comparablehealthy sample of PDH complex, or (ii) contacting the reaction mixturewith a PDH complex activator and comparing an amount of resultantactivation of the PDH complex compared to that of a comparable healthysample of PDH complex.
 24. The method of claim 23(i), wherein the PDHcomplex inhibitor is selected from sodium arsenite and ATP. 25.(canceled)
 26. The method of claim 23(ii), wherein the activator isdichloroactetate.
 27. A kit for use in the method of claim 16, the kitcomprising an antibody specific for said PDH complex
 28. (canceled) 29.The method of claim 16, further comprising: separating remaining samplecontents from the immunocomplex prior to detecting the level ofphosphorylation of immunocomplexed PDH complex.
 30. The method of claim29, wherein the level of phosphorylation is compared by measuring anamount of negative isoelectric point shift of the immunocomplexed PDHcomplex compared to the isoelectric point of the standard, the amount ofnegative isoelectric point shift being directly proportional to theamount of phosphorylation of the PDH complex in the sample.
 31. Themethod of claim 30, wherein the sample is derived from a patient andwherein the amount of negative isoelectric shift is used to screen thepatient for a disorder of PDH complex activity.
 32. The method of claim31, wherein the disorder is a disorder of energy production orutilization.
 33. The method of claim 32, wherein the disorder isdiabetes.
 34. A method for screening to detect an active agent thatmodifies inhibitor or activator activity of a known inhibitor oractivator of PDH complex comprising: a) contacting a sample containingPDH complex in the presence of a known inhibitor or activator and a testactive agent with a PDH complex immunoprecipitating antibody underconditions that allow formation of an antibody/PDH compleximmunocomplex; and b) determining the degree to which the test activeagent modifies the inhibitor or activator activity of the knowninhibitor or activator in the sample as compared to inhibitor oractivator activity of the known inhibitor or activator in the absence ofthe test active agent, thereby detecting an active agent that modifiesinhibitor or activator activity of a known inhibitor or activator of PDHcomplex.
 35. (canceled)
 36. The method of claim 34, wherein: (i) the PDHcomplex inhibitor is sodium arsenite or ATP and the test active agentdecreases inhibitor activity of the PDH complex inhibitor; or (ii) thePDH complex activator is dichloroactetate and the test active agentdecreases activator activity of the PDH complex activator. 37.(canceled)
 38. The method of claim 34, wherein the antibody is ananti-E2 specific antibody, a monoclonal antibody, or a monoclonalanti-E2 specific antibody. 39-40. (canceled)
 41. A method for screeningpatients to identify patients suspected of having a late onsetmitochondrial disorder, said method comprising: a) contacting isolatedantibodies that immunoprecipitate PDH complex with a patient samplecomprising solubilized PDH complex so that the antibodies bind tosolubilized PDH complex present in the sample to form an immunocomplex;b) separating the immunocomplex from the remaining sample contents; andc) detecting a decrease in the amount of PDH complex as compared with anamount in a corresponding normal sample, wherein the decrease indicatesthe patient is suspected of having the late onset mitochondrialdisorder.
 42. The method of claim 41, wherein the late onsetmitochondrial disorder is selected from late onset diabetes,Huntington's, Parkinson's and Alzheimer's diseases, ALS (amyotrophiclateral sclerosis), and Schizophrenia.
 43. The method of claim 41,wherein the separating comprises: i) releasing the immunocomplex; andii) separating the immunocomplex from other components of the sampleusing SDS-PAGE.
 44. The method of claim 41, wherein the anti-PDH complexantibodies are attached to a solid support and the antibodies are taggedwith a detectable marker.
 45. The method of claim 44, wherein thedetecting comprises: (i) contacting the immunocomplex with a detectablemarker that binds specifically to the immunocomplex and measuring theamount of signal from the detectable marker present on the solidsupport, or (ii) high throughput screening.
 46. The method of claim 44,wherein the solid support is beads or a microtiter plate. 47-48.(canceled)